RESUMEN
BACKGROUND: Cytidine deamination that is guided by clustered regularly interspaced short palindromic repeats (CRISPR) can mediate a highly precise conversion of one nucleotide into another - specifically, cytosine to thymine - without generating breaks in DNA. Thus, genes can be base-edited and rendered inactive without inducing translocations and other chromosomal aberrations. The use of this technique in patients with relapsed childhood T-cell leukemia is being investigated. METHODS: We used base editing to generate universal, off-the-shelf chimeric antigen receptor (CAR) T cells. Healthy volunteer donor T cells were transduced with the use of a lentivirus to express a CAR with specificity for CD7 (CAR7), a protein that is expressed in T-cell acute lymphoblastic leukemia (ALL). We then used base editing to inactivate three genes encoding CD52 and CD7 receptors and the ß chain of the αß T-cell receptor to evade lymphodepleting serotherapy, CAR7 T-cell fratricide, and graft-versus-host disease, respectively. We investigated the safety of these edited cells in three children with relapsed leukemia. RESULTS: The first patient, a 13-year-old girl who had relapsed T-cell ALL after allogeneic stem-cell transplantation, had molecular remission within 28 days after infusion of a single dose of base-edited CAR7 (BE-CAR7). She then received a reduced-intensity (nonmyeloablative) allogeneic stem-cell transplant from her original donor, with successful immunologic reconstitution and ongoing leukemic remission. BE-CAR7 cells from the same bank showed potent activity in two other patients, and although fatal fungal complications developed in one patient, the other patient underwent allogeneic stem-cell transplantation while in remission. Serious adverse events included cytokine release syndrome, multilineage cytopenia, and opportunistic infections. CONCLUSIONS: The interim results of this phase 1 study support further investigation of base-edited T cells for patients with relapsed leukemia and indicate the anticipated risks of immunotherapy-related complications. (Funded by the Medical Research Council and others; ISRCTN number, ISRCTN15323014.).
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Inmunoterapia Adoptiva , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Niño , Femenino , Humanos , Antígenos CD19 , Antígenos CD7 , Antígeno CD52 , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunoterapia Adoptiva/efectos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T/genética , Recurrencia , Trasplante de Células Madre , Linfocitos TRESUMEN
Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Glicoproteínas/inmunología , Activación de Linfocitos/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Antígenos CD/genética , Antígenos de Neoplasias/genética , Autoantígenos/inmunología , Antígeno CD52 , Femenino , Citometría de Flujo , Glicoproteínas/genética , Homeostasis/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosforilación/inmunología , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Tirosina Quinasa ZAP-70/inmunologíaRESUMEN
At present, the magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as CD4, LNGFR (low affinity nerve growth factor receptor), and MHC class I molecule H-2Kk. The disadvantage of such markers is the possibility of their undesired and poorly predictable expression by unmodified cells before or after cell manipulation, which makes it essential to develop new surface markers that would not have such a drawback. Earlier, modified CD52 surface protein variants with embedded HA and FLAG epitope tags (CD52/FLAG and CD52/HA) were developed by the group of Dr. Mazurov for the fluorescent cell sorting of CRISPR-modified cells. In the current study, we tested whether these markers can be used for the magnetic selection of transduced cells. For this purpose, appropriate constructs were created in MigR1-based bicistronic retroviral vectors containing EGFP and DsRedExpress2 as fluorescent reporters. Cytometric analysis of the transduced NIH 3T3 cell populations after magnetic selection evaluated the efficiency of isolation and purity of the obtained populations, as well as the change in the median fluorescence intensity (MFI). The results of this study demonstrate that the surface markers CD52/FLAG and CD52/HA can be effectively used for magnetic cell selection, and their efficiencies are comparable to that of the commonly used LNGFR marker. At the same time, the significant advantage of these markers is the absence of HA and FLAG epitope sequences in cellular proteins, which rules out the spurious co-isolation of negative cells.
Asunto(s)
Antígeno CD52 , Proteínas Recombinantes de Fusión , Animales , Ratones , Células 3T3 NIH , Antígeno CD52/metabolismo , Antígeno CD52/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Humanos , Citometría de Flujo/métodos , Separación Celular/métodos , BiomarcadoresRESUMEN
In the past, our research group was able to successfully remove circulating tumor cells with magnetic nanoparticles. While these cancer cells are typically present in low numbers, we hypothesized that magnetic nanoparticles, besides catching single cells, are also capable of eliminating a large number of tumor cells from the blood ex vivo. This approach was tested in a small pilot study in blood samples of patients suffering from chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm. Cluster of differentiation (CD) 52 is a ubiquitously expressed surface antigen on mature lymphocytes. Alemtuzumab (MabCampath®) is a humanized, IgG1κ, monoclonal antibody directed against CD52, which was formerly clinically approved for treating chronic lymphocytic leukemia (CLL) and therefore regarded as an ideal candidate for further tests to develop new treatment options. Alemtuzumab was bound onto carbon-coated cobalt nanoparticles. The particles were added to blood samples of CLL patients and finally removed, ideally with bound B lymphocytes, using a magnetic column. Flow cytometry quantified lymphocyte counts before, after the first, and after the second flow across the column. A mixed effects analysis was performed to evaluate removal efficiency. p < 0.05 was defined as significant. In the first patient cohort (n = 10), using a fixed nanoparticle concentration, CD19-positive B lymphocytes were reduced by 38% and by 53% after the first and the second purification steps (p = 0.002 and p = 0.005), respectively. In a second patient cohort (n = 11), the nanoparticle concentration was increased, and CD19-positive B lymphocytes were reduced by 44% (p < 0.001) with no further removal after the second purification step. In patients with a high lymphocyte count (>20 G/L), an improved efficiency of approximately 20% was observed using higher nanoparticle concentrations. A 40 to 50% reduction of B lymphocyte count using alemtuzumab-coupled carbon-coated cobalt nanoparticles is feasible, also in patients with a high lymphocyte count. A second purification step did not further increase removal. This proof-of-concept study demonstrates that such particles allow for the targeted extraction of larger amounts of cellular blood components and might offer new treatment options in the far future.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Nanopartículas de Magnetita , Humanos , Alemtuzumab/uso terapéutico , Proyectos Piloto , Antígenos CD , Antígeno CD52 , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Neoplasias , Glicoproteínas , Linfocitos , Carbono , Anticuerpos AntineoplásicosRESUMEN
Anti-CD52 antibody (anti-CD52-Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti-CD52-Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS). Our data show that blood and splenic innate immune cells exhibited significantly increased expression of MHC-II and costimulatory molecules, which was associated with increased capacity of activating antigen-specific T cells, at first day but not three weeks after five daily treatment with anti-CD52-Ab in comparison with controls. In contrast to the periphery, microglia and infiltrating macrophages in the CNS exhibited reduced expression levels of MHC-II and costimulatory molecules after antibody treatment at both time-points investigated when compared to controls. Furthermore, the transit response of peripheral innate immune cells to anti-CD52-Ab treatment was also observed in the lymphocyte-deficient SCID mice, suggesting the changes are not a direct consequence of the mass depletion of lymphocytes in the periphery. Our study demonstrates a dynamic and tissue-specific modulation of the innate immune cells in their phenotype and function following the antibody treatment. The findings of differential modulation of the microglia and infiltrating macrophages in the CNS in comparison with the innate immune cells in the peripheral organs support the CNS-specific beneficial effect of alemtuzumab treatment on inhibiting neuroinflammation in multiple sclerosis patients.
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Encefalomielitis Autoinmune Experimental , Alemtuzumab/farmacología , Animales , Anticuerpos , Antígeno CD52/metabolismo , Sistema Nervioso Central/metabolismo , Humanos , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones SCIDRESUMEN
Lymphocyte depletion using anti-CD52 antibody effectively reduces relapses of multiple sclerosis (MS). To begin to understand what mechanisms might control this outcome, we examined the effect of a murine-CD52-specific mAb on the depletion and repopulation of immune cells in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We tested whether the tolerance-promoting receptor programmed cell death protein-1 (PD-1) is required for disease remission post anti-CD52, and found that PD-1-deficient mice with a more severe EAE were nevertheless effectively treated with anti-CD52. Anti-CD52 increased the proportions of newly generated T cells and double-negative (DN) T cells while reducing newly generated B cells; the latter effect being associated with a higher expression of CD52 by these cells. In the longer term, anti-CD52 caused substantial increases in the proportion of newly generated lymphocytes and DN T cells in mice with EAE. Thus, the rapid repopulation of lymphocytes from central lymphoid organs post anti-CD52 may limit further disease. Furthermore, these data identify DN T cells, a subset with immunoregulatory potential, as a significant hyperrepopulating subset following CD52-mediated depletion.
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Linfocitos B/inmunología , Antígeno CD52/antagonistas & inhibidores , Encefalomielitis Autoinmune Experimental/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Alemtuzumab/farmacología , Animales , Antígeno CD52/inmunología , Ratones , Receptor de Muerte Celular Programada 1RESUMEN
BACKGROUND: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. RESULTS: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. CONCLUSION: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.
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Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Isoantígenos/genética , Linfocitos T/metabolismo , Antígeno CD52/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Electroporación , Edición Génica/métodos , Genómica , Células HEK293 , Humanos , Mutación INDEL , PlásmidosRESUMEN
Follicular lymphoma (FL) is a low-grade B-cell malignancy that transforms into a highly aggressive and lethal disease at a rate of 2% per year. Perfect isolation of the malignant B-cell population from a surgical biopsy is a significant challenge, masking important FL biology, such as immune checkpoint coexpression patterns. To resolve the underlying transcriptional networks of follicular B-cell lymphomas, we analyzed the transcriptomes of 34 188 cells derived from 6 primary FL tumors. For each tumor, we identified normal immune subpopulations and malignant B cells, based on gene expression. We used multicolor flow cytometry analysis of the same tumors to confirm our assignments of cellular lineages and validate our predictions of expressed proteins. Comparison of gene expression between matched malignant and normal B cells from the same patient revealed tumor-specific features. Malignant B cells exhibited restricted immunoglobulin (Ig) light chain expression (either Igκ or Igλ), as well the expected upregulation of the BCL2 gene, but also downregulation of the FCER2, CD52, and major histocompatibility complex class II genes. By analyzing thousands of individual cells per patient tumor, we identified the mosaic of malignant B-cell subclones that coexist within a FL and examined the characteristics of tumor-infiltrating T cells. We identified genes coexpressed with immune checkpoint molecules, such as CEBPA and B2M in regulatory T (Treg) cells, providing a better understanding of the gene networks involved in immune regulation. In summary, parallel measurement of single-cell expression in thousands of tumor cells and tumor-infiltrating lymphocytes can be used to obtain a systems-level view of the tumor microenvironment and identify new avenues for therapeutic development.
Asunto(s)
Linfoma de Células B/genética , Linfoma Folicular/genética , Linfocitos T Reguladores/citología , Biopsia , Proteínas Potenciadoras de Unión a CCAAT/genética , Linfocitos T CD4-Positivos/citología , Antígeno CD52/genética , Linaje de la Célula , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Sistema Inmunológico , Inmunoglobulina G , Lectinas Tipo C/genética , Leucocitos Mononucleares/citología , Linfoma de Células B/sangre , Linfoma Folicular/sangre , Tonsila Palatina/metabolismo , Receptores de IgE/genética , Análisis de Secuencia de ARN , Transcriptoma , Microambiente Tumoral , Microglobulina beta-2/genéticaRESUMEN
Anti-cluster of differentiation 52 (CD52) monoclonal antibody (mAb) has been employed in the treatment of chronic lymphoblastic leukemia and multiple sclerosis. Previously we developed a perfusion process to produce the biosimilar mAb named "Mab-TH." A series of quality assessments was conducted in the fields of structural identification, purity analysis, and activity measurement. After these quality researches, this report laid emphasis on preclinical pharmacology and toxicology evaluation. Mab-TH was characterized in biological, pharmacological, and toxicological properties in comparison with the original drug, alemtuzumab. Binding activity and immune-dependent toxicity as in vitro activity were evaluated. Severe immunodeficient mice transplanted with a human leukemia cell line were also used as an in vivo pharmacological model and a 4-week repeated dosing study in cynomolgus monkeys was conducted to evaluate the safety differences. Our results demonstrated that Mab-TH, the anti-CD52 antibody generated by a perfusion process, had high similarity in in vitro and in vivo activities compared with alemtuzumab in relevant preclinical models. The results supported it as a biosimilar candidate for clinical evaluation.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales , Animales , Antígenos CD , Antígenos de Neoplasias , Antígeno CD52 , Diferenciación Celular , Fermentación , Glicoproteínas , Ratones , PerfusiónRESUMEN
CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.
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Antígeno CD52/inmunología , Proteína HMGB1/inmunología , Lectinas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Secuencias de Aminoácidos , Anticuerpos/farmacología , Femenino , Proteína HMGB1/antagonistas & inhibidores , Humanos , Masculino , Dominios Proteicos , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunologíaRESUMEN
Allogeneic haematopoietic stem cell transplantation (HSCT) after a reduced-intensity conditioning (RIC) regimen with fludarabine, melphalan and alemtuzmab is an effective therapy for haematological malignancies. Alemtuzumab, a monoclonal antibody against CD52, a glycosylphosphatidylinositol-anchor-bound surface protein on lymphocytes, depletes T cells to prevent graft-versus-host disease (GVHD). Despite this, acute and chronic GVHD (a/cGVHD) remain life-threatening complications after HSCT. The aim of the present study was to identify parameters to predict GVHD. In 69 patients after HSCT, T-cell subsets were functionally analysed. Reconstitution of CD52neg T cells and CD52neg regulatory T cells (Tregs) correlated with onset, severity and clinical course of aGVHD. Patients with aGVHD showed significantly lower levels of CD52pos T cells compared to patients with cGVHD or without GVHD (P < 0·001). Analysis of T-cell reconstitution revealed a percentage of <40% of CD52pos CD4pos T cells or CD52pos Tregs at day +50 as a risk factor for the development of aGVHD. In contrast, CD52neg Tregs showed significant decreased levels of glycoprotein A repetitions predominant (GARP; P < 0·001), glucocorticoid-induced TNFR-related protein (GITR; P < 0·001), chemokine receptor (CXCR3; P = 0·023), C-C chemokine receptor type 5 (CCR5; P = 0·004), but increased levels of immunoglobulin-like transcript 3 (ILT3; P = 0·001), as well as a reduced suppressive capacity. We conclude that reconstitution of CD52neg T cells and CD52neg Tregs is a risk factor for development of aGVHD.
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Alemtuzumab/administración & dosificación , Antígeno CD52/sangre , Enfermedad Injerto contra Huésped/sangre , Trasplante de Células Madre Hematopoyéticas , Linfocitos T Reguladores/metabolismo , Acondicionamiento Pretrasplante , Enfermedad Aguda , Adulto , Anciano , Aloinjertos , Femenino , Humanos , Masculino , Proteínas de la Membrana/sangre , Persona de Mediana Edad , Receptores CCR5/sangre , Receptores CXCR3/sangre , Factores de RiesgoAsunto(s)
Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Glicoproteínas/inmunología , Activación de Linfocitos/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Animales , Antígeno CD52 , Femenino , Humanos , MasculinoRESUMEN
OBJECTIVE: Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. METHODS: We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing for PIGA mutational analyses. RESULTS: We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of the PIGA gene. One patient with relapsed T-PLL exhibited a single PIGA mutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three different PIGA mutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealed PIGA wild-type sequence reads in all 16 patients with samples available for testing. CONCLUSION: These data indicate that PIGA mutations were acquired during or after completion of alemtuzumab treatment.
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Alemtuzumab/farmacología , Antígeno CD52/genética , Leucemia Prolinfocítica de Células T/genética , Proteínas de la Membrana/genética , Mutación , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Alemtuzumab/uso terapéutico , Antígeno CD52/metabolismo , Análisis Mutacional de ADN , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunofenotipificación , Leucemia Prolinfocítica de Células T/tratamiento farmacológico , Leucemia Prolinfocítica de Células T/metabolismo , Proteínas de la Membrana/metabolismo , Fenotipo , Linfocitos T/patologíaRESUMEN
Alemtuzumab (ALM) is used for T cell depletion in the context of allogeneic hematopoietic stem cell transplantation (alloSCT) to prevent acute graft-versus-host disease and graft rejection. Following ALM-based T cell-depleted alloSCT, relatively rapid recovery of circulating T cells has been described, including T cells that lack membrane expression of the GPI-anchored ALM target Ag CD52. We show, in a cohort of 89 human recipients of an ALM-based T cell-depleted alloSCT graft, that early lymphocyte reconstitution always coincided with the presence of large populations of T cells lacking CD52 membrane expression. In contrast, loss of CD52 expression was not overt within B cells or NK cells. We show that loss of CD52 expression from the T cell membrane resulted from loss of GPI anchor expression caused by a highly polyclonal mutational landscape in the PIGA gene. This polyclonal mutational landscape in the PIGA gene was also found in CD52- T cells present at a low frequency in peripheral blood of healthy donors. Finally, we demonstrate that the GPI-/CD52- T cell populations that arise after ALM-based T cell-depleted alloSCT contain functional T cells directed against multiple viral targets that can play an important role in immune protection early after ALM-based T cell-depleted transplantation.
Asunto(s)
Alemtuzumab/farmacología , Antígeno CD52/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Mutación/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Adulto , Linfocitos B/inmunología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Células Asesinas Naturales/inmunología , Depleción Linfocítica/métodos , Tasa de MutaciónRESUMEN
Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells ("proliferative fraction") in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression. Loss of CD20 can occur in CLL after treatment with rituximab (rtx) and other CD20-targeting mAbs. To broaden the benefit of atz and rtx, we engineered an IgG1-like biAb, atzâ¯×â¯rtx scFv-Fc. The Fc fragment of the biAb facilitates purification by Protein A affinity chromatography and supports a longer circulatory half-life. While atzâ¯×â¯rtx scFv-Fc retained both antigen binding specificities, it showed superior binding to CD52+CD20+ B cells compared to CD52+CD20- T cells. Moreover, atzâ¯×â¯rtx scFv-Fc mediated potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and exhibited B-cell depleting but T-cell sparing activities in vivo in a CLL patient-derived xenograft model. B-cell depletion was more pronounced for cells of the proliferative fraction.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD20/inmunología , Antígeno CD52/antagonistas & inhibidores , Inmunoterapia , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Alemtuzumab , Animales , Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno CD52/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/terapia , Ratones , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adult B-lymphoblastic leukemia (B-ALL) is a hematological malignancy characterized by genetic heterogeneity. Despite successful remission induction with classical chemotherapeutics and novel targeted agents, enduring remission is often hampered by disease relapse due to outgrowth of a pre-existing subclone resistant against the treatment. In this study, we show that small glycophosphatidylinositol (GPI)-anchor deficient CD52-negative B-cell populations are frequently present already at diagnosis in B-ALL patients, but not in patients suffering from other B-cell malignancies. We demonstrate that the GPI-anchor negative phenotype results from loss of mRNA expression of the PIGH gene, which is involved in the first step of GPI-anchor synthesis. Loss of PIGH mRNA expression within these B-ALL cells follows epigenetic silencing rather than gene mutation or deletion. The coinciding loss of CD52 membrane expression may contribute to the development of resistance to alemtuzumab (ALM) treatment in B-ALL patients resulting in the outgrowth of CD52-negative escape variants. Additional treatment with 5-aza-2'-deoxycytidine may restore expression of CD52 and revert ALM resistance.
Asunto(s)
Linfocitos B/metabolismo , Antígeno CD52/deficiencia , Metilación de ADN/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Silenciador del Gen , Glicosilfosfatidilinositoles/deficiencia , Proteínas de la Membrana/genética , Proteínas de Neoplasias/deficiencia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Alemtuzumab/uso terapéutico , Antimetabolitos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Linfocitos B/patología , Antígeno CD52/biosíntesis , Antígeno CD52/genética , Línea Celular Tumoral , Decitabina/farmacología , Decitabina/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genéticaRESUMEN
Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Membrana Celular/inmunología , Epítopos/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales de Origen Murino/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD20/genética , Antígenos CD20/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígeno CD52 , Línea Celular Tumoral , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Inmunoterapia , Ratones , Fagocitosis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Hyperosmolality has been commonly investigated due to its effects on the production and quality characteristics of monoclonal antibodies (mAbs) produced in CHO cell fed-batch cultures. However, the application of hyperosmolality at different times and its effect on biopotency have seldom been researched, especially in perfusion culture. In our study, different degrees of hyperosmolality induced by sodium chloride were investigated in anti-IgE rCHO cell fed-batch cultures and anti-CD52 rCHO cell perfusion cultures during the initial and stable phases. The results showed that the initial hyperosmolality group (IHG) in fed-batch and early phase of perfusion cultures exhibited significant suppression of the viable cell density yet an enhancement in specific productivity, whereas the stable hyperosmolality group (SHG) achieved higher mAb production in both fed-batch and perfusion cultures. Additionally, the SHG produced less aggregates and acidic charge variants than IHG in fed-batch culture, which differed from perfusion cultures. However, the contents of non-glycosylation heavy chain (NGHC) and man5 were higher in SHG than in IHG in fed-batch cultures at plus 60 and 120 mOsm/kg, which was similar to perfusion cultures. Furthermore, the biopotency in the IHG was higher than in the SHG at plus 60 and 120 mOsm/kg in fed-batch cultures, which is similar to complement-dependent cytotoxicity (CDC) efficacy in perfusion cultures. The biopotency of all group was acceptable, except FI3. Thus, the study shows that hyperosmolality at a certain level could be beneficial for both mAb production, quality and biopotency, which could play an important role in process development for commercial production.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Técnicas de Cultivo Celular por Lotes/métodos , Antígeno CD52/inmunología , Inmunoglobulina E/biosíntesis , Concentración Osmolar , Cloruro de Sodio/análisis , Animales , Reactores Biológicos/microbiología , Células CHO , Línea Celular , Proliferación Celular/efectos de los fármacos , Cricetinae , CricetulusRESUMEN
Context: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-guluronic acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive effects in various investigations.Objective: Present research aimed to study the potency of G2013 on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in PBMCs of MS patients under in vitro conditions. Materials and methods: 24 blood samples from MS patients and healthy controls were considered for RT-PCR and flow cytometry techniques under two different doses of G2013.Results: Our research indicated that this drug could significantly decrease the gene expression of TLR2, TLR4 and TNF-α compared to untreated group. Conclusion: Data demonstrated that the guluronic acid is able to modify the expression levels of TLR2, TLR4 and TNF-α genes to less than the pathogenic boarder line level, which it might be recommended for reducing the pathological process in multiple sclerosis.
Asunto(s)
Antígeno CD52/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Hexurónicos/farmacología , Esclerosis Múltiple/metabolismo , Factor 88 de Diferenciación Mieloide/biosíntesis , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 4/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/patologíaRESUMEN
The cell-surface glycoprotein CD52 is widely expressed in lymphocytes. CD4+CD52hi T cells are functioning suppressor CD4+T cells. We investigated the role of the immune regulation of CD4+CD52 T cells in systemic lupus erythematosus (SLE). CD4+CD52lo T cells were increased in SLE patients, in positive correlation with SLEDAI, anti-ds-DNA antibody, and IgG concentration. Circulating follicular helper-like T cells (Tfh-like cells) were also increased in SLE, in positive correlation with CD4+CD52lo T cells. Chemokine receptor 8 (CCR8) expression in CD4+CD52lo T cells was increased. In vitro experiments using CD4 T cells of SLE patients showed that thymus and activation-regulated chemokine (TARC), a ligand of CCR8, contributed to the development of CD4+CD52hi T cells into CD4+CD52lo T cells. Our findings suggest that CD4+CD52lo T-cell upregulation is involved in the production of pathogens by autoantibodies, and TARC may contribute to the development of SLE through an aberrant induction of CD4+CD52lo T cells.